Method and system for microfluidic device and imaging thereof

ABSTRACT

A method for producing an image of an object within a chamber of a microfluidic device includes providing the microfluidic device having x, y, and z dimensions and a chamber depth center point located along the z dimension. The chamber depth center point is located a known z dimension distance from a fiducial marking embedded within the microfluidic device. The method also includes placing the microfluidic device within an imaging system that includes an optical device capable of detecting the fiducial marking. The optical device defines an optical path axially aligned with the z dimension and has a focal plane perpendicular to the optical path. When the focal plane is moved along the optical path, the fiducial marking is maximally detected when the focal plane is at the z depth in comparison to when the focal plane is not substantially in-plane with the z depth.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/645,396, filed Dec. 22, 2009, which is a divisional of U.S.application Ser. No. 10/851,777, filed May 20, 2004, which claimspriority to U.S. Provisional Application Nos. 60/472,226, filed May 20,2003; 60/490,666, filed Jul. 28, 2003; and 60/490,584, filed Jul. 28,2003, all of which are incorporated herein by reference in theirentirety for all purposes.

BACKGROUND OF THE INVENTION

According to the present invention, techniques for microfluidic systems,including a microfluidic chip or circuit, are provided. Moreparticularly, the invention provides a microfluidic structure and methodof manufacture, and a system and method for imaging a microfluidicdevice. Merely by way of example, the fiducial markings are used forprocessing and imaging a microfluidic chip, but it would be recognizedthat the invention has a much broader range of applicability.

Microfluidic techniques have progressed overtime. Certain techniques ofproducing microelectromechanical (MEMS) structures have been proposed.Such MEMS structures include pumps and valves. The pumps and valves areoften silicon-based and are made from bulk micro-machining (which is asubtractive fabrication method whereby single crystal silicon islithographically patterned and then etched to form three-dimensionalstructures). The pumps and valves also use surface micro-machining(which is an additive method where layers of semiconductor-typematerials such as polysilicon, silicon nitride, silicon dioxide, andvarious metals are sequentially added and patterned to makethree-dimensional structures). Unfortunately, certain limitations existwith these conventional MEMS structures and techniques for making them.

As merely an example, a limitation of silicon-based micro-machining isthat the stiffness of the semiconductor materials used necessitates highactuation forces, which result in large and complex designs. In fact,both bulk and surface micro-machining methods are often limited by thestiffness of the materials used. Additionally, adhesion between variouslayers of the fabricated device is also a problem. For example, in bulkmicro-machining, wafer bonding techniques must be employed to createmultilayer structures. On the other hand, when surface micro-machining,thermal stresses between the various layers of the device limits thetotal device thickness, often to approximately 20 microns. Using eitherof the above methods, clean room fabrication and careful quality controlare required.

Accordingly, techniques for manufacturing microfluidic systems using anelastomeric structure have been proposed. As merely an example, thesestructures are often made by forming an elastomeric layer on top of amicromachined mold. The micromachined mold has a raised protrusion whichforms a recess extending along a bottom surface of the elastomericlayer. The elastomeric layer is bonded to other elastomeric layers toform fluid and control regions. The elastomeric layer has overcomecertain limitations of conventional MEMS based structures. Furtherdetails of other characteristics of these elastomeric layers formicrofluidic applications such as crystallization have been providedbelow.

Crystallization is an important technique to the biological and chemicalarts. Specifically, a high-quality crystal of a target compound can beanalyzed by x-ray diffraction techniques to produce an accuratethree-dimensional structure of the target. This three-dimensionalstructure information can then be utilized to predict functionality andbehavior of the target.

In theory, the crystallization process is simple. A target compound inpure form is dissolved in solvent. The chemical environment of thedissolved target material is then altered such that the target is lesssoluble and reverts to the solid phase in crystalline form. This changein chemical environment is typically accomplished by introducing acrystallizing agent that makes the target material less soluble,although changes in temperature and pressure can also influencesolubility of the target material.

In practice however, forming a high quality crystal is generallydifficult, often requiring much trial and error and patience on the partof the researcher. Specifically, the highly complex structure of evensimple biological compounds means that they are usually not amenable toforming a highly ordered crystalline structure. Therefore, a researcherneeds to be patient and methodical, experimenting with a large number ofconditions for crystallization, altering parameters such as sampleconcentration, solvent type, countersolvent type, temperature, andduration in order to obtain a high quality crystal.

A high-throughput system for screening conditions for crystallization oftarget materials, for example proteins, is provided in a microfluidicdevice. The array of metering cells is formed by a multilayerelastomeric manufacturing process. Each metering cell comprises one ormore of pairs of opposing chambers, each chamber being in fluidcommunication with the other through an interconnecting microfluidicchannel, one chamber containing a protein solution, and the other,opposing chamber, containing a crystallization reagent. Along thechannel, a valve is situated to keep the contents of opposing chambersfrom each other until the valve is opened, thus allowing free interfacediffusion to occur between the opposing chambers through theinterconnecting microfluidic channel. As the opposing chambers approachequilibrium with respect to crystallization reagent and proteinconcentrations as free interface diffusion progresses, the protein wouldat some point, form a crystal under certain conditions. In someembodiments, the microfluidic devices taught by Hansen et al. are havearrays of metering cells containing chambers for conducting proteincrystallization experiments therein. Use of such arrays in turn providesfor high-throughput testing of numerous conditions for proteincrystallization which require analysis. See PCT publication WO02/082047, published Oct. 17, 2002 and by Hansen, et al. PCT publicationWO 02/082047 is incorporated by reference herein in its entirety for allpurposes.

From the above, it is seen that improved techniques for elastomericdesign and analysis are highly desirable.

BRIEF SUMMARY OF THE INVENTION

According to the present invention, techniques for microfluidic systems,including a microfluidic chip or circuit, are provided. Moreparticularly, the invention provides a microfluidic structure and methodof manufacture, and a system and method for imaging a microfluidicdevice. Merely by way of example, the fiducial markings are used forprocessing and imaging a microfluidic chip, but it would be recognizedthat the invention has a much broader range of applicability.

In a specific embodiment, the invention provides a biological substrate,e.g., microfluidic chip. The substrate includes a rigid substratematerial, which has a surface region capable of acting as a handlesubstrate. The substrate also has a deformable fluid layer (e.g.,polymeric material, silicone, silicone rubber, rubber, plastic, PDMS)coupled to the surface region. One or more well regions are formed in afirst portion of the deformable fluid layer and are capable of holding afluid therein. The one or more channel regions are formed in a secondportion of the deformable fluid layer and are coupled to one or more ofthe well regions. An active region is formed in the deformable fluidlayer. Such active region includes the one or more well regions, whichare designed to hold fluid. A non-active region is formed in thedeformable fluid layer. The non-active region is formed outside of thefirst portion and the second portion. Preferably, at least threefiducial markings are formed within the non-active region and disposedin a spatial manner associated with at least one of the well regions. Acontrol layer is coupled to the fluid layer. Preferably, the substratealso includes an other fiducial marking with pre-designed shape andsize, including at least an edge and center region.

In an alternative specific embodiment, the invention provides a methodof fabricating a biological substrate. The method includes providing arigid substrate material, which has a surface region and is capable ofacting as a handle substrate. The method includes coupling a deformablefluid layer to the surface region of the rigid substrate. The deformablelayer has one or more well regions formed in a first portion of thedeformable fluid layer and one or more channel regions formed in asecond portion of the deformable fluid layer. An active region is formedin the deformable fluid layer. A non-active region is formed in thedeformable fluid layer and is formed outside of the first portion andthe second portion. Preferably, at least three fiducial markings areformed within the non-active region and are disposed in a spatial mannerassociated with at least one of the well regions. The method alsoincludes coupling a control layer to the fluid layer.

In yet an alternative embodiment, the invention provides a method ofmanufacturing microfluidic chip structures. The method includesproviding a mold substrate including a plurality of well patterns. Eachof the well patterns is provided within a portion of an active region ofa fluidic chip. The method includes forming a plurality of fiducialmarking patterns around a vicinity of each of the well patterns. Each ofthe plurality of fiducial marking patterns is within a portion of anon-active region of a fluidic chip. The plurality of fiducial markingpatterns includes a set of alignment marks disposed spatially aroundeach of the well patterns. The method also includes forming a thicknessof deformable material within the plurality of well patterns and withinthe plurality of fiducial marking patterns to fill a portion of the moldsubstrate. The method includes coupling the thickness of deformablematerial including a plurality of wells formed from the well patternsand a plurality of fiducial marking patterns formed from the fiducialmarking patterns to rigid substrate material.

In yet an alternatively embodiment, the present invention provides amicrofluidic system. The system has a rigid substrate material, whichincludes a surface region that is capable of acting as a handlesubstrate. The system has a deformable fluid layer coupled to thesurface region. One or more well regions is formed in a first portion ofthe deformable fluid layer. The one or more well regions is capable ofholding a fluid therein. The system has one or more channel regionsformed in a second portion of the deformable fluid layer. The one ormore channel regions is coupled to one or more of the well regions. Anactive region is formed in the deformable fluid layer. The active regionincludes the one or more well regions. A non-active region is formed inthe deformable fluid layer. The non-active region is formed outside ofthe first portion and the second portion. A first fiducial marking isformed within the non-active region and is disposed in a spatial mannerassociated with at least one of the channel regions. A second fiducialmarking is formed within the non-active region and is disposed in aspatial manner associated with at least one of the well regions. Acontrol layer is coupled to the fluid layer. The control layer includesone or more control regions. A third fiducial marking is formed withinthe control layer.

In yet an alternative specific embodiment, the present inventionprovides another microfluidic system. The system has a substratecomprising a surface region. A deformable layer is coupled to thesurface of the substrate. The deformable layer comprises at least athickness of first material. A control layer is coupled to thedeformable layer to form a sandwich structure including at least thesubstrate, the deformable layer and the control layer. The control layeris made of at least a thickness of second material. At least onefiducial marking is provided within either the control layer or thedeformable layer or the substrate. The fiducial marking is characterizedby a visual pattern provided in a volume surrounded wholly or partiallyby at least the substrate, the first material, or the second material.Preferably, a fluid is disposed within the open volume of the onefiducial marking. The fluid is characterized by a refractive index thatis substantially lower than its surrounding regions, e.g., firstthickness of material, second thickness of material, substrate. That is,the refractive index may be associated with air or other like fluid andthe surrounding regions are characterized by a refractive indexassociated with a solid according to a specific embodiment.

Numerous benefits are achieved using the present invention overconventional techniques. The invention provides at least one way to formalignment patterns for a deformable active region for a microfluidicsystem according to a specific embodiment. The invention can also useconventional materials, which are relatively easy to use. Preferably,the invention provides at least two sets of alignment marks, includingone set of spatially disposed fiducial markings and a pre-designatedpattern, which has an edge and center region. Depending upon theembodiment, one or more of these benefits may exist. These and otherbenefits have been described throughout the present specification andmore particularly below.

In yet another specific embodiment, the invention provides a method forprocessing a microfluidic device, e.g., microfluidic chip, biologicalchip. The method includes providing a flexible substrate including afirst plurality of fiducial markings, and determining a first pluralityof actual locations corresponding to the first plurality of fiducialmarkings respectively. The first plurality of fiducial markings isassociated with a first plurality of design locations respectively.Additionally, the method includes processing information associated withthe first plurality of actual locations and the first plurality ofdesign locations, and determining a transformation between a designspace and a measurement space. The design space is associated with thefirst plurality of design locations, and the measurement space isassociated with the first plurality of actual locations. Moreover, themethod includes performing a first alignment to the flexible substratebased on at least information associated with the transformation betweenthe design space and the measurement space. Also, the method includesacquiring a first plurality of images of the first fiducial marking,processing information associated with the first plurality of images,performing a second alignment to the flexible substrate based on atleast information associated with the first plurality of images, andacquiring a second image of the flexible substrate.

According to yet another embodiment, a method for processing amicrofluidic device includes providing a flexible substrate including atleast three fiducial markings, a first additional fiducial marking, anda first chamber capable of holding a fluid therein. Additionally, themethod includes determining a transformation between a design space anda measurement space based on at least information associated with the atleast three fiducial markings, and performing a first alignment to theflexible substrate based on at least information associated with thetransformation between the design space and the measurement space.Moreover, the method includes acquiring at least a first image of thefirst additional fiducial marking associated with the first chamber,performing a second alignment to the flexible substrate based on atleast information associated with the first image, and acquiring asecond image of the first chamber associated with the flexiblesubstrate.

According to yet another embodiment, the invention provides a system forprocessing one or more microfluidic devices. The system includes one ormore computer-readable media and a stage for locating a flexiblesubstrate. The flexible substrate comprises at least three fiducialmarkings, a first additional fiducial marking, and a first chambercapable of holding a fluid therein. The one or more computer-readablemedia include one or more instructions for providing a flexiblesubstrate, and one or more instructions for determining a transformationbetween a design space and a measurement space based on at leastinformation associated with the at least three fiducial markings.Additionally, the one or more computer-readable media include one ormore instructions for performing a first alignment to the flexiblesubstrate based on at least information associated with thetransformation between the design space and the measurement space, oneor more instructions for acquiring at least a first image of the firstadditional fiducial marking associated with the first chamber, one ormore instructions for performing a second alignment to the flexiblesubstrate based on at least information associated with the first image,and one or more instructions for acquiring a second image of the firstchamber associated with the flexible substrate.

According to yet another embodiment of the present invention, a methodfor processing a microfluidic device includes providing a flexiblesubstrate (e.g., polymer, silicone based, rubber) comprising one or morewell regions and a plurality of fiducial marks. The well regions arecapable of holding a fluid therein and at least three of the fiducialmarks are within a vicinity of one of the well regions. Preferably, theflexible substrate has been provided on a rigid member. The methodincludes locating the flexible substrate on a stage and capturing animage of at least the three fiducial marks within the vicinity of theone well region of the flexible substrate to generate a mapping from adesign space to a measurement space. The method also includes aligningthe flexible substrate to an image acquisition location using at leastthe mapping from the design space and one additional fiducial mark,wherein the at least one additional fiducial mark is associated with theone well region. The method also includes acquiring a high-resolutionimage of at least the one well region and storing the high-resolutionimage in a memory.

In yet another alternative specific embodiment, the invention provides asystem for processing one or more microfluidic devices. The systemincludes one or more computer memories. The system also includes a stagefor locating a flexible substrate, which has one or more well regionsand a plurality of fiducial marks. The well regions are capable ofholding a fluid therein. At least three of the fiducial marks are withina vicinity of one of the well regions. The one or more computer memoriescomprise one or more computer codes. The one or more computer codesinclude a first code directed to capturing an image of at least thethree fiducial marks within the vicinity of the one well region of theflexible substrate to generate a mapping from a design space to ameasurement space. A second code is directed to aligning the flexiblesubstrate to an image acquisition location using at least the mappingfrom the design space and one additional fiducial mark, wherein the atleast one additional fiducial mark is associated with the one wellregion. A third code is directed to acquiring a high-resolution image ofat least the one well region. A fourth code is directed to storing thehigh-resolution image in a memory. Depending upon the embodiment, theremay also be other computer codes to implement the functionalitydescribed herein as well as outside of the specification.

In yet another alternative specific embodiment, the invention providesmethod of processing a biological microfluidic device. The methodincludes providing a deformable substrate comprising one or moremetering cells, which are capable of containing a fluid therein. Themethod also includes locating the deformable substrate on a stagetranslatable in x, y, and z directions and translating the stage toimage at least four fiducial marks associated with the deformablesubstrate. The method determines x, y, and z positions (or other likespatial positions) of the at least four fiducial marks according to apreferred embodiment. The method computes a non-planar mapping between adesign space and a measurement space based on the x, y, and z positionsof the at least four fiducial marks and translates the stage to an imageacquisition position calculated using the non-planar mapping. A step ofcapturing an image of at least one metering cell is included.

According to yet another embodiment of the present invention, a methodfor producing an image of an object within a chamber of a microfluidicdevice includes providing the microfluidic device. The microfluidicdevice has x, y, and z dimensions and a chamber depth center pointlocated between a top wall and a bottom wall of the chamber along the zdimension. The chamber depth center point is located a known z dimensiondistance from an optically detectable fiducial marking embedded withinthe microfluidic device at a z depth. Additionally, the method includesplacing the microfluidic device within an imaging system. The imagingsystem includes an optical device capable of detecting the fiducialmarking and transmitting the image of the object. The optical devicedefines an optical path axially aligned with the z dimension of themicrofluidic device and has a focal plane perpendicular to the opticalpath. When the focal plane is moved along the optical path in line withthe fiducial marking, the fiducial marking is maximally detected whenthe focal plane is at the z depth in comparison to when the focal planeis not substantially in-plane with the z depth. Additionally, theimaging system includes an image processing device in communication withthe optical device. The image processing device is able to control theoptical device to cause the focal plane to move along the z axis andmove the focal plane to maximally detect the fiducial marking. The imageprocessing device is further able to transmit the image of the object.Additionally, the method includes controlling the optical device withthe image processing device to cause the focal plane to move along theoptical path until the optical device maximally detects the fiducialmarking. Moreover, the method includes controlling the optical devicewith the image processing device to move the focal plane along theoptical path the z dimension distance to cause the field depth centerpoint to be located at the chamber depth center point. Moreover, themethod includes imaging the object within the chamber while the focalplane is located at the chamber depth center point.

According to yet another embodiment of the present invention, a systemfor producing an image of an object within a chamber of a microfluidicdevice includes the microfluidic device. The microfluidic device has x,y, and z dimensions and a chamber depth center point located between atop wall and a bottom wall of the chamber along the z dimension. Thechamber depth center point is located a known z dimension distance froma optically detectable fiducial marking embedded within the microfluidicdevice at a z depth. Additionally, the system includes an imaging systemfor placing the microfluidic device therein. The imaging system includesan optical device capable of detecting the fiducial marking andtransmitting the image of the object. The optical device defines anoptical path axially aligned with the z dimension of the microfluidicdevice and having a focal plane. When the focal plane is moved along theoptical path in line with the fiducial marking, the fiducial marking ismaximally detected when the focal plane is substantially in-plane withthe z depth as compared to when the field depth center point is notsubstantially in-plane with the z depth. Additionally, the imagingsystem includes an image processing device in communication with theoptical device. The image processing device is able to control theoptical device to cause the focal plane to move along the z axis andmove the field depth center point to maximally detect the fiducialmarking. The image processing device is able to transmit the image ofthe object. The image processing device is in operable communicationwith the optical device to cause the focal plane to move along theoptical path until the optical device maximally detects the fiducialmarking. When the image processing device causes the optical device tomove the focal plane along the optical path the z dimension distance,the focal point is located at said chamber depth center point.

According to yet another embodiment of the present invention, a methodfor producing an image of a chamber within a microfluidic deviceincludes imaging the microfluidic device to produce an image using animaging system having an optical path in the z plane of the microfluidicdevice, and mapping from the image a first set of coordinates of themicrofluidic device to determine whether the microfluidic device isskewed or distorted when compared to a coordinate map of an idealmicrofluidic device. Additionally, the method includes positioning themicrofluidic device so as to position the chamber within the opticalpath based on a matrix transformation calculated coordinate positiondetermined by computing a matrix transformation between the first set ofcoordinates of the microfluidic device and the coordinate map of theideal microfluidic device. Moreover, the method includes obtaining atime zero image of the microfluidic device chamber. The time zero imagecontains images of artifacts present in the microfluidic device. Also,the method includes obtaining a second image of the microfluidic devicechamber and subtracting the first image of the microfluidic devicechamber from the second image of the microfluidic chamber to produce animage of the chamber without time zero artifacts.

Numerous benefits are achieved using the present invention overconventional techniques. Some embodiments provide alignment and/or focusbased on mapping between the design space and the measurement space. Thetransformation between the design space and the measurement space uses,for example, at least three fiducial markings. Certain embodimentsprovide accurate focusing by acquiring and analyzing a plurality ofimages along at least one dimension. Some embodiments of the presentinvention perform alignment and focusing on a microfluidic deviceincluding at least one flexible substrate. The alignment and focusingtake into account the deformation of the flexible substrate. Certainembodiments improve throughput in imaging system. For example, theimaging system uses a computer system to automatically perform alignmentand focusing. In another example, mapping from the design space to themeasurement space increases the accuracy of stage positioning, andthereby, the efficiency of high-resolution image acquisition. Dependingupon the embodiment, one or more of these benefits may exist. These andother benefits have been described throughout the present specificationand more particularly below.

Various additional objects, features and advantages of the presentinvention can be more fully appreciated with reference to the detaileddescription and accompanying drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-10 are simplified diagrams illustrating a method for fabricatinga microfluidic system according to an embodiment of the presentinvention;

FIG. 11 is a simplified cross-sectional view diagram of a microfluidicsystem according to an embodiment of the present invention;

FIG. 12 is a simplified top-view diagram of a microfluidic systemaccording to an alternative embodiment of the present invention;

FIG. 13 is a simplified top and side-view diagram of a microfluidicsystem according to an alternative embodiment of the present invention;

FIG. 13A is a simplified top-view diagram of a microfluidic systemincluding carrier and identification code according to an embodiment ofthe present invention;

FIG. 14 is a simplified imaging system for imaging objects within amicrofluidic device according to an embodiment of the present invention;

FIGS. 15A and 15B are a simplified microfluidic device according to anembodiment of the present invention;

FIGS. 16A and 16B are simplified actual image in measurement space andsimplified virtual image in design space respectively according to anembodiment of the present invention;

FIGS. 17A, 17B, and 17C show a simplified method for image subtractionand masking according to an embodiment of the present invention;

FIG. 18 is a simplified imaging method for microfluidic device accordingto an embodiment of the present invention;

FIG. 19 is a simplified method for mapping between the measurement spaceand the design space according to an embodiment of the presentinvention;

FIG. 20 is a simplified diagram for fiducial markings according to anembodiment of the present invention;

FIG. 21 is a simplified method for locating fiducial marking accordingto an embodiment of the present invention;

FIG. 22 is a simplified metering cell shifted from design positionaccording to an embodiment of the present invention;

FIG. 23 is a simplified method for aligning and focusing image systemaccording to an embodiment of the present invention;

FIG. 24 is a simplified method for acquiring images of fiducial markingaccording to an embodiment of the present invention;

FIG. 25 is a simplified method for aligning and focusing image systemaccording to an embodiment of the present invention;

FIG. 26 is a simplified image acquired and analyzed according to anembodiment of the present invention;

FIG. 27 shows simplified curves for focus score as a function of zposition obtained at the process 804 according to an embodiment of thepresent invention;

FIG. 28 shows simplified curves for focus score as a function of zposition according to one embodiment of the present invention;

FIG. 29 shows simplified curves for focus score as a function of zposition according to another embodiment of the present invention; and

FIG. 30 is a simplified surface map of a three dimensional flexiblesubstrate according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, techniques for microfluidic systems,including a microfluidic chip or circuit, are provided. Moreparticularly, the invention provides a microfluidic structure and methodof manufacture, and a system and method for imaging a microfluidicdevice. Merely by way of example, the fiducial markings are used forprocessing and imaging a microfluidic chip, but it would be recognizedthat the invention has a much broader range of applicability.

Method for Manufacturing Fluidic Chip

A method for manufacturing a fluidic chip according to an embodiment ofthe present invention may be outlined below. Certain details of themethod 100 are also provided according to a flow diagram illustrated byFIG. 1, which is not intended to unduly limit the scope of the claimsherein. One of ordinary skill in the art would recognize manyvariations, alternatives, and modifications.

1. Form a mold substrate for a moldable channel and well layer 101,including fiducial markings;

2. Form molded channel and well layers 102, including fiducial markings,overlying the mold substrate via spinning of silicone material;

3. Form a mold substrate for a moldable control layer 103;

4. Form molded control layer overlying the mold substrate via spinningof silicone material 104;

5. Align molded channel and well layers overlying the molded controllayer 105;

6. Remove molded channel and well layers from the mold substrate for themolded channel and well layers to form a sandwiched structure includingthe channel and well layers and control layer 106;

7. Align the channel and well layers to a transparent substrate surface107;

8. Bond the sandwiched structure including the aligned channel and welllayers to the transparent substrate 108;

9. Provide the sandwiched structure for use in a fluidic processingsystem 109; and

10. Perform other steps 110, as desired.

The above sequence of steps provides a method for manufacturing amicrofluidic system having molded channel, well, and control layers. Ina specific embodiment, each of the molded channel, well, and controllayers is deformable or elastic. That is, well regions may vary slightlyfrom well to well throughout a single microfluidic system, which hasbeen provided on a chip. To compensate for such deformablecharacteristic, the present system includes at least one or morefiducial markings that have been placed in predetermined spatiallocations to be used with image processing techniques. These fiducialmarkings allow for any inherent errors caused by the deformablecharacteristic to be compensated at least in part using the imageprocessing techniques. Further details of methods and resultingstructures of the present microfluidic system have been describedthroughout the present specification and more particularly below.

Method for Manufacturing Mold for Fluid Layer

A method for manufacturing a mold for a fluid layer according to anembodiment of the present invention may be outlined below. Certaindetails of the method 200 are also provided according to a flow diagramillustrated by FIG. 2, which is not intended to unduly limit the scopeof the claims herein. One of ordinary skill in the art would recognizemany variations, alternatives, and modifications.

1. Provide mold substrate material 201;

2. Apply first layer of photoresist onto mold substrate 202;

3. Pattern including fiducials (e.g., dots) the first layer ofphotoresist to form channel regions 203;

4. Form channel regions including fiducials through the patterned filmon the mold substrate material 204;

5. Strip first layer of photoresist 205;

6. Apply second layer of photoresist 206;

7. Align pattern onto the second layer of photoresist based upon one ormore of the channel regions 207;

8. Pattern including wells, x-marks, and company logo aligned tochannels (where alignment is provided by matching brackets) the secondlayer of photoresist 208;

9. Form channels, x-marks, and company logo through the patterned secondfilm on the mold substrate material 209;

10. Strip second layer of photoresist to form completed mold substratematerial including channel and well structures 210; and

11. Perform other steps, as desired.

The above sequence of steps provides a method for manufacturing a moldfor a molded channel and well layers according to a specific embodiment.In a specific embodiment, each of the molded channel and well layers isdeformable or elastic. To compensate for such deformable characteristic,the present system includes at least one or more fiducial markings thathave been placed in predetermined spatial locations to be used withimage processing techniques. These fiducial markings allow for anyinherent errors caused by the deformable characteristic to becompensated at least in part using the image processing techniques.Further details of methods and resulting structures of the presentmicrofluidic system have been described throughout the presentspecification and more particularly below.

Method for Manufacturing Control Layer

A method for manufacturing a mold for a control layer according to anembodiment of the present invention may be outlined below. Certaindetails of the method 300 are also provided according to a flow diagramillustrated by FIG. 3, which is not intended to unduly limit the scopeof the claims herein. One of ordinary skill in the art would recognizemany variations, alternatives, and modifications.

1. Provide mold substrate material 301;

2. Apply first layer of photoresist onto mold substrate 302;

3. Pattern the first layer of photoresist to form control fluid regions303;

4. Form control fluid regions through the patterned film on the moldsubstrate material 304;

5. Strip first layer of photoresist to form completed mold substratematerial including control fluid regions 305; and

6. Perform other steps, as desired.

The above sequence of steps provides a method for manufacturing a moldfor a molded control layer according to a specific embodiment. In aspecific embodiment, the control layers is deformable or elastic. Tocompensate for such deformable characteristic, the present systemincludes at least one or more fiducial markings that have been placed inpredetermined spatial locations to be used with image processingtechniques. These fiducial markings allow for any inherent errors causedby the deformable characteristic to be compensated at least in partusing the image processing techniques. Further details of methods andresulting structures of the present microfluidic system have beendescribed throughout the present specification and more particularlybelow.

FIGS. 1-11 are simplified diagrams illustrating a method for fabricatinga microfluidic system according to an embodiment of the presentinvention. These diagrams are merely examples, which should not undulylimit the scope of the claims herein. One of ordinary skill in the artwould recognize many variations, modifications, and alternatives. Asnoted above, FIGS. 1 through 3 have been described. Certain featureswith regard to illustrating features of the fluidic system have beenprovided by way of FIGS. 4 through 11. For easy viewing, the left sideillustrates an overview of the entire substrate, including patterns,while the right side illustrates a portion of the pattern that ispertinent according to a feature being described.

Referring to FIG. 4, fluid channel layer is illustrated. The fluidchannel layer (or control layer) includes fluid channels 401 to deliverfluid throughout the substrate 403. Fiducial markings in shape ofcircles 405 are used to locate the channels themselves. These circlesare part of the fluid channel layer mask and are transferred with thechannels onto the substrate. The circles are recessed regions, which donot extend all the way through the layer, in preferred embodiments.

Referring to FIG. 5, a well layer 501 including well regions 501, 503 onthe substrate are illustrated. The well layer includes the well regionsand the company logo 507 (which serves as a predetermined fiducialmarking according to preferred embodiments) that enables x-y spatiallocation of a metering cell. In addition the logo is also used forfocusing onto the wells as the logo height is the same height as thewells. The well layer also includes a plurality of fiducial markings505, e.g., crosses. Such crosses are located within a vicinity of eachof the well regions. The crosses are at a finite distance and aretranslated from the mask to the substrate. When using image processingalgorithms to locate one or more of the wells, the crosses can be usedas a reference to well location. As shown, each of the crosses arelocated in a spatial manner around a periphery of the well region. Thatis, each of the crosses occupies a corner region that is not active andis free from the well itself.

Referring to FIG. 6, alignment occurs between the fluid channel layerand well layer according to a specific embodiment. Here, the methodaligns these two layers at the substrate mold making process. The welllayer has a different thickness and shape than the fluid layer. The welllayer produces sharp edges while fluid channel layer produces roundedges. Preferably, a goal is to have the wells overlaying the channelsin order for the channels to distribute fluids into the wells. The welllayer mask is aligned to the fluid layer to place wells over the fluidchannels, as shown. Alignment is done by matching the frame of the welllayer to the frame of the fluid channel layer.

The method generally forms more than one design 701 on a substratematerial as shown in FIG. 7. Each of these designs can be separatedusing regions 703 according to a preferred embodiment. The methodperforms final assembly after silicone (or other like material) has beenpoured separately over the fluid/well layer mold and the control layermold. Preferably, the final assembly is made when the control layer ofsilicone is aligned to the fluid layer of silicone. Matching alignmentmarks are located on the fluid and control layer that need to overlayeach other for proper alignment.

To align the patterned substrate to the blank substrate, the methodincludes placing a template of the patterned substrate underneath theblank substrate, which is transparent, as illustrated by FIG. 8. Thetemplate allows carrier top access to reagent inputs. In addition properalignment of the patterned substrate onto the blank transparentsubstrate enables the imaging station to view the global fiducials onthe chip through the carrier bottom. As shown, FIG. 9 illustrates thepatterned substrate, including wells and channels, overlying thetransparent substrate. Details of the fiducial markings are providedthroughout the present specification and more particularly below.

FIG. 10 is a simplified top-view diagram 1000 of a completedmicrofluidic system including well 1001 and channel regions 1003. Asshown, fiducial markings 1005 are disposed spatially around a peripheryof the well region. The system also has company log 1007, which is apredetermined fiducial marking, which is larger in size than the otherfiducial markings. The predetermined fiducial marking has one or moreedges and a center region, among other features, as needed. Of course,one of ordinary skill in the art would recognize many other variations,modifications, and alternatives. Specific details with regard to thepresent system are also provided using the side-view diagram illustratedbelow.

FIG. 11 is a simplified cross-sectional view diagram 1115 of amicrofluidic system 1100 according to an embodiment of the presentinvention. This diagram is merely an example, which should not undulylimit the scope of the claims herein. One of ordinary skill in the artwould recognize many variations, modifications, and alternatives. Asshown, the system includes a glass substrate 1103 or any liketransparent substrate material, which can act as a handle substrate.Overlying the handle substrate is fluid channel 1105 and well layer1107. The fluid channel and well layer have been provided on a singlelayer 1109 or can be multiple layers. The fluid channel has a depth thatis less than the well, which extends into the single layer. Preferably,the fluid channel and well layer are made using a suitable material suchas silicone, silicon rubber, rubber, plastic, PDMS, or other polymericmaterial. Preferably, the material is also transparent, but may bedeformable or alternatively flexible in characteristic. The system alsohas a control layer 1111, which includes control channel 1113.Preferably, the control layer is made using a suitable material such assilicone, silicon rubber, rubber, plastic, PDMS, or other polymericmaterial. Depending upon the embodiment, there may also be otherfeatures in the system.

One 1102 of a plurality of fiducial markings is also shown. The markingis at a vicinity of the well region and also has a height relative tothe wells that are substantially similar. That is, optically the heightof the marking is about the same as the well relative to a planeparallel to the substrate. Alternatively, the marking may be formedbased upon a predetermined off-set relative to the plane parallel to thesubstrate in other embodiments. Certain dimension are also shown, butare not intended to be limiting in any manner. Depending upon theembodiment, there can be many variations, alternatives, andmodifications.

Other embodiments of the present invention are provided below.

FIG. 12 is a simplified top-view diagram of a microfluidic systemaccording to an alternative embodiment of the present invention. Thisdiagram is merely an example, which should not unduly limit the scope ofthe claims herein. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives. As shown, the systemcomprises a biological substrate 1200. The substrate includes a rigidsubstrate material, which has a surface region. The substrate is capableof acting as a handle substrate. The rigid substrate can be made of asuitable material such as a glass, a plastic, silicon, quartz,multi-layered materials, or any combination of these, and the like. Ofcourse, the type of substrate used depends upon the application.

The substrate also includes a deformable fluid layer coupled to thesurface region. Preferably, the fluid layer is attached using a gluelayer or other attachment technique. One or more well regions are formedin a first portion of the deformable fluid layer. The one or more wellregions is capable of holding a fluid therein. One or more channelregions is formed in a second portion of the deformable fluid layer, Theone or more channel regions is coupled to one or more of the wellregions. The channel regions include protein channels 1201 and reagentchannels 1203. Other channel regions can also be included.

The fluid layer includes active and non-active regions. An active regionis formed in the deformable fluid layer. The active region includes theone or more well regions. A non-active region is formed in thedeformable fluid layer. The non-active region is formed outside of thefirst portion and the second portion. The term “active” and “non-active”are merely used for illustration purposes and should not limit the scopeof the claims herein. The non-active region generally corresponds toregions free from use of fluids or other transport medium, and the like.

The substrate includes a plurality of fiducial markings. Each of thefiducial markings is selectively placed within a certain layer region.In a specific embodiment, a first fiducial marking 1205 is formed withinthe non-active region and disposed in a spatial manner associated withat least one of the channel regions. That is, the first fiducial markingis within the channel regions. Preferably, the first fiducial marking isa recessed region that includes a selected width and depth. The recessedregion forms a pattern to be captured by an image processing technique.In a specific embodiment, a second fiducial marking 1213 is formedwithin the non-active region and disposed in a spatial manner associatedwith at least one of the well regions. That is, the second fiducialmarking is within the channel regions. Preferably, the second fiducialmarking is a recessed region that includes a selected width and depth.The recessed region forms a pattern to be captured by an imageprocessing technique.

The substrate also has a control layer coupled to the fluid layer. Thecontrol layer includes one or more control regions. The control layerincludes interface control line 1207 and containment control line 1209.Other control lines can also be included. Preferably, a third fiducialmarking 1211 is formed within the control layer. Preferably, the thirdfiducial marking is a recessed region that includes a selected width anddepth. The recessed region forms a pattern to be captured by an imageprocessing technique. Further details of the substrate can be foundthroughout the present specification and more particularly below.

FIG. 13 is a simplified top and side-view diagram 1300 of a microfluidicsystem according to an alternative embodiment of the present invention.This diagram is merely an example, which should not unduly limit thescope of the claims herein. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives. As shown,the diagram includes a “top-view,” a “detailed top view” and “side view”of fluidic microstructures according to embodiments of the presentinvention. As shown, the system also includes global fiducials 1301. Theglobal fiducials are used for rough alignment purposes, although may beused for fine alignment as well. In one embodiment, the global fiducialsby a spatial dimension of greater than 100 μm and less than 250 μm. Forexample, the global fiducials include a length and a width of about 180μm and 160 μm respectively. In another embodiment, the global fiducialsare characterized by a depth of at least 10 μm within a thickness of thenon-active region. For example, the global fiducials include a thicknessof about 20 μm and are within the deformable layer 1305 as shown. Theside view diagram includes a substrate 1302, which is preferably rigid,with an upper surface region. The rigid substrate can be made of asuitable material such as a glass, a plastic, silicon, quartz,multi-layered materials, or any combination of these, and the like. Ofcourse, the type of substrate used depends upon the application.

The substrate also includes a deformable fluid layer coupled to thesurface region. Preferably, the fluid layer is attached using a gluelayer or other attachment technique. One or more well regions are formedin a first portion of the deformable fluid layer. The one or more wellregions 1309 is capable of holding a fluid therein. As shown, the wellregion has a certain thickness within the deformable layer. One or morechannel regions 1311 is formed in a second portion of the deformablefluid layer. The one or more channel regions is coupled to one or moreof the well regions. The channel regions include protein channels andreagent channels. Other channel regions can also be included. As shown,the channel regions are not as thick as the well regions. The deformablelayer includes an upper surface, which couples to control layer 1307. Asshown, the control layer includes a plurality of control channels 1313.

Fiducial markings are selectively placed in a spatial manner on themicrofluidic system. In a specific embodiment, the global alignmentfiducial marking is formed in the deformable layer within a vicinity ofa well region. A first fiducial marking is placed within a vicinity ofthe well region. In one embodiment, four wells form a metering cell. Themetering cell has a length and a width each about 2 μm. The firstfiducial marking is placed substantially at the center of the meteringcell. A second fiducial marking may be placed within a vicinity of thechannel region within the deformable layer. A third fiducial marking maybe placed within a vicinity of the control channel in the control layer.Depending upon the application, there may be variations, alternatives,and modifications. That is, two of the fiducial markings may be within avicinity of the channel region and the third fiducial marking may bewithin a vicinity of the control channel in the control layer.Alternatively, two of the fiducial markings may be within a vicinity ofthe well region and the third fiducial marking may be within a vicinityof the control channel in the control layer. Preferably, the fiducialmarkings are placed within a vicinity of the region being examined, suchas well or channel regions. The fiducial marking placed within thecontrol layer or another layer serves as an alignment point to correctfor depth of field or other optical characteristics.

As shown in FIGS. 1-13, various fiducial markings can be included inmicrofluidic systems. In one embodiment, preferably a fiducial markingcomprises a recessed region in the deformable layer. The recessed regionbecomes a volume or open region surrounded by portions of the deformablelayer or other layers. The volume or open region is preferably filledwith a fluid such as a gas including air or other non-reactive fluid.The fluid also has a substantially different refractive index to lightrelative to the surrounding deformable layer. The open region ispreferably filed with an air or air type mixture and has a lowrefractive index. Similarly, the fiducial marking in the control layerhas similar characteristics according to a specific embodiment. Incertain embodiments, the fiducial marking has sharp edges that highlightthe marking from its surroundings. For example, the edges are preferably90 degree corners or the like. Of course, one of ordinary skill in theart would recognize other variations, modifications, and alternatives.

Additionally, as shown in FIGS. 1-13, the fluid channel and well layerare made using a suitable material such as silicone, silicon rubber,rubber, plastic, PDMS, or other polymeric material in certainembodiments. The control layer can be made also using a suitablematerial such as silicone, silicon rubber, rubber, plastic, PDMS, orother polymeric material in some embodiments. In other embodiments, thefluid channel and well layer and the control layer are made of material,whose thermal coefficient is at least 10⁻⁴. For example, the thermalcoefficient ranges from 10⁻⁴ to 10⁻³. In yet another example, thethermal coefficient equals about 3×10⁻³. In yet other embodiments, thefluid channel and well layer and the control layer are made of material,whose Young's modulus is at most 5×10⁶. For example, the Young's modulusranges from 8×10⁴ to 7.5×10⁵.

Also, as shown in FIGS. 1-13, the microfluidic device includes thechannel regions and well regions. These diagram are merely examples,which should not unduly limit the scope of the claims. One of ordinaryskill in the art would recognize many variations, alternatives, andmodifications. In certain embodiments, the channel regions and the wellregions are interchangeable. The channels and the wells refer torecessed regions in the microfluidic device. In other embodiments, themicrofluidic device uses channel regions to function as well regions. Inyet other embodiments, the microfluidic device includes chambers thatcan be used as fluid channels, control channels, and wells.

FIG. 13A is a simplified top-view diagram of a microfluidic systemincluding carrier and identification code according to an embodiment ofthe present invention. This diagram is merely an example, which shouldnot unduly limit the scope of the claims herein. One of ordinary skillin the art would recognize other variations, modifications, andalternatives. As shown, a system 1350 includes a chip 1353, which hasassociated carrier 1351. The chip can be any one of the embodimentsreferred to as a microfluidic system herein as well as others. The chipgenerally includes a substrate, deformable layer, and control layer,among other features. The chip also has well regions coupled to channelregions in the deformable layer. The control layer is coupled to thedeformable layer. The carrier includes various features such asinlets/outlets 1355 that couple to elements in the chip. The carrieralso includes accumulation reservoirs 1357, which couple to theinlets/outlets. The carrier has an identification region 1358 thatincludes barcode or other identification element. Other identificationfeatures, which can be identified visually, may also be used. Furtherembodiments may also include other identification devices such as radiofrequency identification, pattern recognition, and the like.

Preferably, the bar code is an encoded set of lines and spaces ofdifferent widths that can be scanned and interpreted into numbers toidentify certain features of the microfluidic system. The barcodeincludes intrinsic and/or extrinsic information associated with thechip. The intrinsic information may be pattern recognition informationand/or alignment information associated with the fiducial markings. Thatis, once identification and alignment of the system has occurred usingat least the fiducial markings, such alignment information can be storedin memory of a computing or processing system according to an embodimentof the present invention. The alignment information can be used to moreefficiently process the specific chip, including bar code, for certainapplications. The alignment information associated with the fiducialmarkings can be stored in memory that is later retrievable usingprocessing systems according to embodiments of the present invention.Further details of these processing systems can be found throughout thepresent specification and more particularly below.

FIG. 14 is a simplified imaging system for imaging objects within amicrofluidic device according to an embodiment of the present invention.This diagram is merely an example, which should not unduly limit thescope of the claims. One of ordinary skill in the art would recognizemany variations, alternatives, and modifications.

As shown in FIG. 14, an imaging system 4010 includes a stage 4020. Thestage 4020 is movable in x, y, and z dimensions, as shown by arrows4190. The movement of the stage 4020 is caused by a stage drive 4025under control of a computer system 4110. Additionally, the imagingsystem 4010 includes an imaging device 4060. The imaging device 4060includes an lens system 4070 with lenses 4075 therein, and a detector4080. The lens system 4070 is under control of the computer system 4110to automatically adjust the focus of the lens system 4070 in response toimage information gathered by the detector 4080. The image iscommunicated to the computer system 4110 and stored in a database 4115.

The lens system 4070 can focus on a microfluidic device 4030 byadjusting a focal plane 4100 in the z direction. For example, the focalplane is at a chamber centerline of the microfluidic device 4030. Themicrofluidic device 4030 is situated upon the stage 4020 and can havevarious structures. For example, the microfluidic device has a structureand is manufactured by a method as described in FIGS. 1-13. In anotherexample, the microfluidic device 4030 has a chamber 4050 wherein anobject, such as a protein crystal, may be formed or otherwise located.For example, the chamber 4050 is capable to hold a volume of fluid lessthan 1 nanoliter. A plurality of chambers can be combined to form ametering cell. The chamber 4050 has a chamber centerline that is locatedbetween a top wall and a bottom wall of the chamber 4050. For example,the chamber 4050 is a well region, a channel region, or both.

Moreover, the imaging system 10 includes an illumination device 4170 forproducing an illumination beam 4180. For example, the illumination beam4180 illuminates objects within the microfluidic device 4030.Additionally, the computer system 4110 may be in communication with aninput/output device 4160 and a barcode reader 4120. The barcode reader4120 can read a bar code 4130 on a microfluidic device 4140. Forexample, the microfluidic device 4140 is used as the microfluidic device4030.

Although the above has been shown using a selected group of apparatusesfor the system 4010, there can be many alternatives, modifications, andvariations. For example, some of the apparatuses may be expanded and/orcombined. Other apparatuses may be inserted to those noted above.Depending upon the embodiment, the arrangement of apparatuses may beinterchanged with others replaced. Further details of these apparatusesare found throughout the present specification.

For example, the imaging system 4010 may be integrated into a largerrobotic system, such as a rotating arm or railroad track type roboticsystem, to increase the throughput. The imaging system 4010 cancommunicate with the robotic system and control the flow of microfluidicdevices into and out of the imaging system, acquire information aboutthe microfluidic devices and their contents, and supply image data andresults from the imaging system to the robotic system. If the roboticsystem includes a database, the imaging system can contribute image andresults to the database. The robotic system, in-turn, may automaticallydesign further experiments based upon the results provided by theimaging system.

According to an embodiment of the present invention, the imaging system4010 operates in the following manner including a plurality ofprocesses. These processes are merely examples, which should not undulylimit the scope of the claims. One of ordinary skill in the art wouldrecognize many variations, alternatives, and modifications. Themicrofluidic device 4030 is securely placed on the stage 4020. Based ona fixed feature of the microfluidic device 4030, the computer system4110 instructs the drive 4025 to move the stage 4020 and align themicrofluidic device 4030 with a first fiducial marking. For example, thefiducial marking is embedded within the microfluidic device 4030 at aknown z dimension distance from the chamber centerline. In anotherexample, the first fiducial marking comes into focus by the imagingdevice 4060 based on dead reckoning from the fixed feature. The actualcoordinates of the first fiducial marking is then measured andregistered with the imaging system 4010. Additionally, the actualcoordinates of two or more additional fiducial markings are measured andregistered.

The actual locations of the fiducial markings are compared with theirdesign locations in the stored image map respectively. For example, thestored image map is associated with the design space. In anotherexample, the stored image map is an ideal image map. In yet anotherexample, the stored image map is associated with a mathematical grid.Based on the comparison, the imaging system 4010 determines whetherstretch, distortion, or other deformation exists in the microfluidicdevice 4030. If differences are present between the actual fiduciallocations and the design fiducial locations, a matrix transformation,such as an Affine transformation, is performed. The transformationconverts the actual shape of a metering cell into a virtual shape withrespect to the design space. By converting the actual image to thevirtual image, an image subtraction and other image analysis may beperformed.

Although the above has been shown using a selected sequence of processesfor operating the imaging system 4010, there can be many alternatives,modifications, and variations. For example, some of the processes may beexpanded and/or combined. Other processes may be inserted to those notedabove. Depending upon the embodiment, the specific sequences of stepsmay be interchanged with others replaced. Further details of theseprocesses are found throughout the present specification.

FIGS. 15A and 15B are a simplified microfluidic device according to anembodiment of the present invention. These processes are merelyexamples, which should not unduly limit the scope of the claims. One ofordinary skill in the art would recognize many variations, alternatives,and modifications. FIGS. 15A and 15B depict a top view and across-sectional view of a microfluidic device respectively. Amicrofluidic device 4230 includes at least a flexible substrate with achamber 4250 and a fiducial marking 4254. For example, the fiducialmarkings 4254 are used for xyz alignment and focus of an imaging system.In one embodiment, the imaging system focuses upon the fiducial markings4254 within the microfluidic device 4230 and conduct mapping between themeasurement space and the design space. The imaging system then adjustsa focal plane with respect to the z dimension of the microfluidic device4230 and places the focal plane in plane with a selected point withinthe chamber 4250, preferably at chamber focus position 4256. The chamberfocus position 4256 is a Δz distance 4252 away from a focus plane 4258of the fiducial markings 4254. For example, at the focus plane 4258, thefiducial markings 4254 are optimally focused. In one embodiment, themicrofluidic device 4230 may be used as the microfluidic device 4030. Inanother embodiment, the microfluidic device may be made by processesdescribed in FIGS. 1-13A.

FIGS. 16A and 16B are simplified actual image in measurement space andsimplified virtual image in design space respectively according to anembodiment of the present invention. These diagrams are merely examples,which should not unduly limit the scope of the claims. One of ordinaryskill in the art would recognize many variations, alternatives, andmodifications. For example, the design space is ideal, and themeasurement space is distorted.

The difference between the design space and the measurement space can becalculated through fiducial mapping. Consequently, a matrixtransformation is developed to convert the actual image into a virtualimage in the design space. Transforming various actual images into thesame design space facilitates the image subtraction and masking in orderto maximize the viewable area of a metering cell chamber. Moreover, if adefect or debris is present within the chamber at time zero in a seriesof time based images, such defect or debris can be masked out ofsubsequent images to avoid false positive when applying automatedcrystal recognition analysis. Additionally, the walls of a chamber maybe subtracted from subsequent images to reduce the likelihood of falsereading in the crystal recognition analysis.

FIGS. 17A, 17B, and 17C show a simplified method for image subtractionand masking according to an embodiment of the present invention. Thesediagrams are merely examples, which should not unduly limit the scope ofthe claims. One of ordinary skill in the art would recognize manyvariations, alternatives, and modifications.

FIG. 17A depicts a metering cell with debris, shown as the letter “D”distributed about the metering cell chambers. The metering cell istransformed into the design space. For example, the metering cell isrotated to align with the design coordinate system and stretchcompensated to make the metering cell dimensions match those of thedesign metering cell dimensions. The foreign objects not present in thedesign metering cell are masked out such that the regions including andimmediately surrounding the foreign objects are masked. The masking canreduce the likelihood of falsely triggering the crystal detectionanalysis into deeming the foreign objects as crystals that were formed.FIG. 17B depicts a masked image where the foreign objects have beenmasked.

Additionally, the walls in FIG. 17A can be removed by image subtraction.FIG. 17C depicts an image without chamber walls. From FIG. 17C, furthermasking may be performed if wall implosion is detected. The wallimplosion may occur when the microfluidic device is dehydrating and thechamber contents are permeating outside of the chamber, causing anegative pressure therein and thus wall collapse or implosion. Suchfurther masking for implosion may employ a series of known shapes thatoccur when chamber implosion occurs and uses such known shapes to createadditional masks to occlude from the image the now intruding implodedwalls.

FIG. 18 is a simplified imaging method for microfluidic device accordingto an embodiment of the present invention. This diagram is merely anexample, which should not unduly limit the scope of the claims. One ofordinary skill in the art would recognize many variations, alternatives,and modifications. The method 4400 includes process 4410 for mappingbetween measurement space and design space, process 4420 for alignmentand focusing, and process 4430 for capturing image. In one embodiment,the method 4400 may be performed by the imaging system 4010 on themicrofluidic device 4030. For example, the imaging system 4010 performsthe processes 4410, 4420, and 4430 according to the instructions of thecomputer system 4110 or another computer system. Although the above hasbeen shown using a selected sequence of processes, there can be manyalternatives, modifications, and variations. For example, some of theprocesses may be expanded and/or combined. Other processes may beinserted to those noted above. For example, a process of placing amicrofluidic device on the stage of an imaging system is performed priorto the process 4410. Depending upon the embodiment, the specificsequences of processes may be interchanged with others replaced. Forexample, the process 4420 may be skipped. Further details of theseprocesses are found throughout the present specification and moreparticularly below.

At the process 4410, the measurement space and the design space aremapped. FIG. 19 is a simplified process 4410 for mapping between themeasurement space and the design space according to an embodiment of thepresent invention. This diagram is merely an example, which should notunduly limit the scope of the claims. One of ordinary skill in the artwould recognize many variations, alternatives, and modifications. Theprocess 4410 includes process 4440 for locating fiducial marking,process 4442 for measuring actual location of fiducial marking, process4444 for comparing actual location and design location of fiducialmarking, process 4446 for determining need for additional fiducialmarking, process 4448 for determining transformation between measurementspace and design space, and process 4450 for coarse alignment. Althoughthe above has been shown using a selected sequence of processes, therecan be many alternatives, modifications, and variations. For example,some of the processes may be expanded and/or combined. Other processesmay be inserted to those noted above. Depending upon the embodiment, thespecific sequences of processes may be interchanged with othersreplaced. In one embodiment, the processes 4440, 4442, and 4444 may beperformed for more than one fiducial markings before the process 4446 isperformed. For example, several fiducial markings are located andmeasured. In another embodiment, the process 4444 may be performed afterthe process 4446 has determined no additional mark needs to located.Further details of these processes are found throughout the presentspecification and more particularly below.

At the process 4440, a fiducial marking is located on a microfluidicdevice. For example, the microfluidic device is the microfluidic device4030. FIG. 20 is a simplified diagram for fiducial markings according toan embodiment of the present invention. This diagram is merely anexample, which should not unduly limit the scope of the claims. One ofordinary skill in the art would recognize many variations, alternatives,and modifications. As shown in FIG. 20, each of fiducial markings 4520,4522, and 4524 includes three plus signs or crosses located at threecorners of a square and a company logo located at the fourth corner ofthe square. The fiducial marking 4520, 4522, or 4524 is the fiducialmarking located at the process 4440. In one example, the fiducialmarking 4520, 4522, or 4524 is a global fiducial. In another example,the fiducial marking 4520, 4522, or 4524 serves as both a globalfiducial and a local fiducial. In yet another example, the fiducialmarking 4520, 4522, or 4524 is located in the same plane as the wellregions of the microfluidic device.

In another embodiment of the present invention, the located fiducialmarking has a configuration different from the fiducial marking 4520,4522, or 4524. In another embodiment, the located fiducial marking isreadily recognizable by the image processing algorithm. Operation of theimage processing algorithm is improved when the fiducial marking isreadily visible, with minimal optical interference from the edge of themicrofluidic device or other channels.

Locating the fiducial marking at the process 4440 can be performedmanually, automatically, or both. For example, the fiducial marking ismoved and identified in the field of view of the imaging system byvisual inspection. In another example, the imaging system automaticallyplaces and identifies the fiducial marking in the field of view.

According to an embodiment of the present invention, FIG. 21 is asimplified process 4440 for locating fiducial marking. This diagram ismerely an example, which should not unduly limit the scope of theclaims. One of ordinary skill in the art would recognize manyvariations, alternatives, and modifications. The process 4440 includesprocess 4810 for acquiring image, process 4820 for segmenting image,process 4830 for performing blob analysis, process 4840 for determiningwhether fiducial marking is located, process 4850 for adjustingposition, and process 4860 for moving fiducial marking. Although theabove has been shown using a selected sequence of processes, there canbe many alternatives, modifications, and variations. For example, someof the processes may be expanded and/or combined. Other processes may beinserted to those noted above. Depending upon the embodiment, thespecific sequences of processes may be interchanged with othersreplaced. For example, the process 4860 may be skipped. Further detailsof these processes are found throughout the present specification andmore particularly below.

At the process 4810, an image of the fiducial marking is acquired. Priorto the process 4810, the stage is positioned to an initial positiondefined as {right arrow over (r)}₀=x₀{circumflex over(x)}+y₀ŷ+z₀{circumflex over (z)}. At the process 4810, an image of thefiducial marking is captures. In one embodiment, the image is capturedby a digital camera such as a Leica DC500. In another embodiment, theimage has a low resolution. For example, the image is 640×480 pixels insize, and the color depth resolution is 16 bits. In another example, thepixel and color depth resolutions are varied to optimize systemperformance. After the image is acquired, the image may be adjusted tocompensate for variations in lamp intensity and color. This compensationmay take the form of image normalization. Additionally, the red, blue,and green components of the image can be adjusted to white balance theimage. The white-balancing of the image may be accomplished by mediancorrection or other known techniques.

At the process 4820, the image is segmented. Segmentation of the imagecan separate desired images from the background signal and produce“blobs” useful in further analysis steps. At the process 4830, the blobanalysis is performed. The blobs in the image are compared against atraining set contained in a database. The training set contains imagesof a fiducial marking obtained from a large number of microfluidicdevices and imaging conditions. For example, the fiducial marking is thecompany logo. In another example, the fiducial marking is one other thanthe company logo.

At the process 4840, whether the fiducial marking is located isdetermined. If the fiducial marking is located, the process 4442 isperformed. In one embodiment, if the best match of the blobs to thestandards is found to be within a predetermined specification, thefiducial marking is considered to be located. For example, thepredetermined specification includes a proximity ranking of less than4200. If the fiducial marking is not detected, the process 4850 isperformed.

At the process 4850, the position of the stage is adjusted. After theadjustment, the processes 4810, 4820, 4830 and 4840 are performed. Inone embodiment, at the process 4850, the stage is moved in an xdirection and/or a y direction. In another embodiment, the stage ismoved in a z direction at the process 4850. For example, the stage ismoved by a selected amount in a first z-direction (Δz) by stepping thez-motor of the stage in a first selected direction. At each steppedz-height, the processes 4810, 4820, 4830 and 4840 are performed. Theprocess 4850 is repeated until the fiducial marking is determined to belocated at the process 4840 or the stage reaches the end of its range ofmotion in the first z direction. If the stage reaches the end of itsrange of motion, the stage is returned to the initial position, {rightarrow over (r)}₀ and the stage is stepped by Δz in a second selectedz-direction. For example, the second z-direction is opposite to thefirst z-direction. The step size Δz can be uniform in both directions,or vary as a function of direction or distance from {right arrow over(r)}₀. At each stepped z-height in the second direction, the processes4810, 4820, 4830, and 4840 are performed. The process 4850 is repeateduntil the fiducial marking is located or the stage reaches the end ofits range of motion in the second z direction. If the fiducial markingcannot be located within the range of motion, an error message isgenerated. In yet another embodiment, at the process 4850, the stage ismoved in an x direction, a y direction, and/or a z direction.

At the process 4860, the stage is translated to move the fiducialmarking to substantially the center of the field of view of the imagingsystem.

As shown in FIG. 19, at the process 4442, the actual location of thelocated fiducial marking is measured. As shown in FIG. 20, the measuredlocation of the fiducial marking 4520 is represented by vector {rightarrow over (r)}_(1m) with respect to the origin O 4510. The measuredvector {right arrow over (r)}_(nm) representing the actual location of afiducial marking can also be written as:

$\begin{matrix}{{\overset{\rightharpoonup}{r}}_{nm} = \begin{bmatrix}x_{nm} \\y_{nm} \\z_{nm}\end{bmatrix}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

where n is a positive integer. For example, the actual location {rightarrow over (r)}_(nm) is automatically detected by an image processingroutine.

At the process 4444, the actual location and the design location of thefiducial marking is compared. The design location of the fiducialmarking 4520, referenced to an origin O, can be represented by a designvector {right arrow over (r)}_(1D). The design vector {right arrow over(r)}_(nD) representing the design location of a fiducial marking canalso be written as:

$\begin{matrix}{{\overset{\rightharpoonup}{r}}_{nD} = \begin{bmatrix}x_{nD} \\y_{nD} \\z_{nD}\end{bmatrix}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$

where n is a positive integer. The difference in the design location{right arrow over (r)}_(nD) and the measured location {right arrow over(r)}_(nm) can be calculated as {right arrow over (r)}_(n0)={right arrowover (r)}_(nD)−{right arrow over (r)}_(nm).

As discussed above and further emphasized here, the processes 4440,4442, and 4444 are only examples. One of ordinary skill in the art wouldrecognize many variations, alternatives, and modifications. In oneembodiment, at the processes 4440, 4442, and 4444, the imaging systemuses a predetermined magnification objective. For example, a 10×magnification objective is used for the lenses 4075 of the imagingsystem 4010.

In another embodiment, the imaging system first uses a lower powermagnification objective, such as a 2.5× magnification objective, at theprocesses 4440, 4442, and 4446. Subsequently, for the same fiducialmarking, the coarse alignment of the microfluidic device is performed.For example, the coarse alignment uses the difference vector {rightarrow over (r)}_(n0). The vector {right arrow over (r)}_(n0) representsthe translation of the located fiducial marking in the x, y, and z axesfrom the design location. Using the x and y scalar values from {rightarrow over (r)}_(n0), the stage position of the imaging system can beadjusted in the x-y plane to position the located fiducial marking at apre-determined location in the x-y plane. Additionally, using the z-axisscalar value from {right arrow over (r)}_(n0), the position of the stagecan be adjusted in the z plane to position the fiducial marking at aselected location in the z plane. The z-axis focus adjustment may beperformed before, after, and/or at the same time as the adjustment inthe x-y plane.

Afterwards, the imaging system switches to a higher power magnificationobjective, for example, a 10× magnification objective. For example, themeasurements and adjustments made with a lower power objective place thefiducial marking within the field of view of the imaging objective whenthe objective is switched to the higher power magnification objective.With the higher power magnification objective, the image system can moreaccurately determine the vectors {right arrow over (r)}_(nm) and {rightarrow over (r)}_(n0).

At the process 4446, whether an additional fiducial marking should belocated and measured is determined. If an additional fiducial markingdoes not need to be located and measured, the process 4448 is performed.If an additional fiducial marking should be located and measured, theprocess 4440 is performed.

For example, the processes 4440, 4442, and 4444 are performed for eachof the three fiducial markings 4520, 4522, and 4524 as shown in FIG. 20.For the fiducial marking 4520, {right arrow over (r)}_(1m) and {rightarrow over (r)}₁₀ are determined. For the fiducial marking 4522, {rightarrow over (r)}_(2m) and {right arrow over (r)}₂₀ are determined. Forthe fiducial marking 4524, {right arrow over (r)}_(3m) and {right arrowover (r)}₃₀ are determined. In other embodiments, more than three globalfiducial markings are located and measured.

At the process 4448, the transformation between measurement space anddesign space is determined. For example, a matrix transformation, suchas an Affine transformation, is determined based on the differencevectors {right arrow over (r)}_(n0).

In one embodiment of the present invention, using a flexiblemicrofluidic device, non-uniform absorption of fluids, non-uniformhydration and dehydration, or other factors, can result in flexing,stretching, shrinking, bowing, swelling, contracting and otherdistortions in the microfluidic device. In addition, fabricationprocesses for the device, handling during packaging and testing, andother protocols can introduce deformations and distortions in thedevice. These deformations may be dimensionally uniform or non-uniform,including both linear and non-linear distortions. The effects of thesedistortions may impact the magnitude and direction of the measuredvectors {right arrow over (r)}_(nm). Accordingly, the deviation of thesemeasured vectors from their corresponding design vectors {right arrowover (r)}_(nD) represent the linear and non-linear distortions of themicrofluidic device media. Using the difference vectors {right arrowover (r)}_(n0), a transformation can be created between the design spaceand the measurement space. This transformation is correlated with theflexing, stretching, bowing, and other distortions and deformationspresent in the microfluidic device. The transformation may have linearcomponents and/or non-linear components.

For example, a transformation is determined based on three fiducialmarkings, such as the fiducial markings 4520, 4522, and 4524. Suchtransformation can provide a planar mapping of the microfluidic device.The plane defined by the three fiducial markings can be used tocharacterize the translation of the microfluidic device in the threedimensions of x, y, and z as well as stretching of the microfluidicdevice material in the plane of the microfluidic device. The roll,pitch, and yaw of this plane can also be characterized by the planedefined by the three fiducial markings.

At the process 4450, the coarse alignment is performed with thetransformation between the design space and the measurement space. Forexample, the actual position of a metering cell of the microfluidicdevice is determined, and the metering cell is positioned in preparationfor imaging. For example, the actual location of a metering cell can beshifted from the design location due to distortions and deformations ofthe microfluidic device. Not only can the plane of the microfluidicdevice be translated and tilted, the microfluidic device can bestretched in the plane of the microfluidic device, further shifting theactual position of the metering cell. In one embodiment, the meteringcell is shifted in the x dimension and/or the y dimension. In anotherembodiment, the metering cell is shifted in the z dimension.

FIG. 22 is a simplified metering cell shifted from design positionaccording to an embodiment of the present invention. This diagram ismerely an example, which should not unduly limit the scope of the claimsherein. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives. In FIG. 22, a metering cell4710 in the design space is schematically illustrated with solid linesand the same metering cell 4730 in the measurement space isschematically illustrated in dashed lines. The design vector {rightarrow over (r)}_(D) points to a design location 4715 of a fiducialmarking of the metering cell 4710, and the measured vector {right arrowover (r)}_(M) points to a design location 4735 of the same fiducialmarking of the same metering cell 4730. The tip of the measured vector{right arrow over (r)}_(M) is offset from the design vector {right arrowover (r)}_(D) by an error vector {right arrow over (r)}_(δ). This errorvector can have components in all three dimensions. Using thetransformation from design space to measurement space, the approximateactual location of a metering cell can be calculated by taking intoaccount the error vector. The stage of the imaging system can be movedin the x dimension, the y dimension, and/or the z dimension to positionthe metering cell in preparation for imaging.

As discussed above, FIG. 19 is merely an example, which should notunduly limit the scope of the claims. One of ordinary skill in the artwould recognize many variations, alternatives, and modifications. Forexample, to initiate the microfluidic device registration process, analgorithm can be used to register the microfluidic device with respectto the coordinates of the imaging system coupled to camera and thestage. Based on determinations and/or evaluations of the stack-uptolerances from the integrated microfluidic device and carrier and themicroscope stage, tolerance metrics can be set. In one embodiment, thetolerances is set to ensure that at least one global fiducial generallyappears within the field of view available when the lenses 4075 comprisea 2.5× objective. This tolerance definition allows automation of thefiducial finding process and streamline system operation. In anotherembodiment, an automated system can locate a fiducial marking outsidethe current field of view of the imaging system through a searchroutine. Additionally, the movement of the fiducial mark can beperformed, for example, by moving the stage with respect to the imagingdevice, moving the imaging device with respect to the stage, or both.The stage carries the microfluidic device to which the fiducial markbelongs.

As shown in FIG. 18, at the process 4420, the alignment and focusing areperformed. FIG. 23 is a simplified process 4420 for aligning andfocusing image system according to an embodiment of the presentinvention. This diagram is merely an example, which should not undulylimit the scope of the claims. One of ordinary skill in the art wouldrecognize many variations, alternatives, and modifications. The process4420 includes process 4802 for acquiring images of and process 4804 fordetermining alignment and focus. Although the above has been shown usinga selected sequence of processes, there can be many alternatives,modifications, and variations. For example, some of the processes may beexpanded and/or combined. Other processes may be inserted to those notedabove. For example, a process substantially similar to the process 4440as described in FIG. 21 is performed on a metering cell and itsassociated fiducial marking, which are aligned, focused and imaged atthe processes 4802 and 4804. Depending upon the embodiment, the specificsequences of processes may be interchanged with others replaced. Furtherdetails of these processes are found throughout the presentspecification and more particularly below.

At the process 4802, images of a fiducial marking is acquired Forexample, the fiducial marking is associated with the metering cell,which has been aligned using the mapping between the design space andthe measurement space at the process 4450. FIG. 24 is a simplifiedprocess 4802 for acquiring images of fiducial marking according to anembodiment of the present invention. This diagram is merely an example,which should not unduly limit the scope of the claims. One of ordinaryskill in the art would recognize many variations, alternatives, andmodifications. The process 4802 includes process 4910 for moving stagein first direction, process 4920 for moving stage in second direction,process 4930 for acquire image, and process 4940 for determining needfor additional movement. Although the above has been shown using aselected sequence of processes, there can be many alternatives,modifications, and variations. For example, some of the processes may beexpanded and/or combined. Other processes may be inserted to those notedabove. Depending upon the embodiment, the specific sequences ofprocesses may be interchanged with others replaced. For example, thefiducial marking used in the process 4802 may be a company logo having aheight the same as that of the wells. In another example, the fiducialmarking has a height different from that of the wells. The known offsetbetween the plane of the fiducial marking and that of the wells wouldenable accurate z-axis adjustments to be made. Further details of theseprocesses are found throughout the present specification and moreparticularly below.

At the process 4910, the stage of the imaging system is moved in a firstz direction. As discussed above, at the process 4450, the metering celland its associated fiducial marking can be aligned in the x dimension,the y dimension, and/or the z dimension based on the transformationbetween the design space and the measurement space. At the end ofprocess 4450, the z position of the stage is referred to as z_(f). Atthe process 4910, the stage is moved from z_(f) by a distance in a firstz-direction equal to z_(f)+δz.

At the process 4920, the stage is moved in a second z-direction by adistance equal to δz. For example, this second z direction is oppositeto the first z direction. The step size δz can be uniform or vary as afunction of distance from z_(f).

At the process 4930, an image of the fiducial marking is acquired. Inone embodiment, the image is captured by a digital camera such as aLeica DC500. In another embodiment, the image has a low resolution. Forexample, the image is 640×480 pixels in size, and the color depthresolution is 16 bits. In another example, the pixel and color depthresolutions are varied to optimize system performance. After the imageis acquired, the image may be adjusted to compensate for variations inlamp intensity and color. This compensation may take the form of imagenormalization. Additionally, the red, blue, and green components of theimage can be adjusted to white balance the image. The white-balancing ofthe image may be accomplished by median correction or other knowntechniques.

At the process 4940, whether additional stage movement should beperformed is determined. If the stage has been moved in the seconddirection though a distance equal to or larger than 2·z₁+δz, noadditional stage movement is needed. The process 4804 should beperformed. If the stage has been moved in the second direction though adistance smaller than 2·z₁+δz, an additional stage movement is needed.The process 4920 is performed.

As shown in FIG. 23, at the process 4804, the alignment and focus aredetermined. FIG. 25 is a simplified process 4804 for aligning andfocusing image system according to an embodiment of the presentinvention. This diagram is merely an example, which should not undulylimit the scope of the claims. One of ordinary skill in the art wouldrecognize many variations, alternatives, and modifications. The process4804 includes 6810 for selecting image, process 6820 for segmentingimage, process 6830 for performing blob analysis, process 6840 fordetermining whether fiducial marking is located, process 6850 fordetermining need for additional image, process 6860 for determiningalignment, process 6870 for determining focus scores, and process 6880for determining focus position. Although the above has been shown usinga selected sequence of processes, there can be many alternatives,modifications, and variations. For example, some of the processes may beexpanded and/or combined. Other processes may be inserted to those notedabove. Depending upon the embodiment, the specific sequences ofprocesses may be interchanged with others replaced. For example, theprocess 6860 is skipped. In another example, the process 6860 isperformed after the process 6880. Further details of these processes arefound throughout the present specification and more particularly below.

At the process 6810, an image is selected from the images taken in theprocess 4802 for further analysis. At the process 6820, the selectedimage is segmented. Segmentation of the image can separate desired imagefrom the background signal and produce “blobs” useful in furtheranalysis steps.

At the process 6830, the blob analysis is performed. The blobs in theimage are compared against a training set contained in a database. Thetraining set contains images of a fiducial marking obtained from a largenumber of microfluidic devices and imaging conditions. For example, thefiducial marking is the company logo. In another example, the fiducialmarking is one other than the company logo.

At the process 6840, whether the fiducial marking is located isdetermined. If the fiducial marking is located, a region of interest(ROI) is created around the fiducial marking FIG. 26 is a simplifiedimage acquired and analyzed according to an embodiment of the presentinvention. This diagram is merely an example, which should not undulylimit the scope of the claims. One of ordinary skill in the art wouldrecognize many variations, alternatives, and modifications. The fiducialmarking may be a company log 4770 surround by a region of interest 4760.In one embodiment, if the best match of the blobs to the standards isfound to be within a predetermined specification, the fiducial markingis considered to be located. For example, the predeterminedspecification includes a proximity ranking of less than 4200.

At the process 6850, whether additional image should be analyzed isdetermined. For example, if any of the images taken at the process 4802has not been selected at the process 6810, the process 6810 is performedto select the image not yet selected. If all of the images taken at theprocess 4802 have been selected, the process 6860 is performed.

At the process 6860, the alignment in the x and y dimensions isdetermined. In one embodiment, the alignment uses the actual location ofan ROI and the design location of the ROI. For example, the alignment inthe x and y dimensions are determined by the difference between theactual location and the design location. In another embodiment, thefiducial marking has a known spatial relationship with chambers withinthe metering cell in the x and y dimensions. The alignment in the x andy dimensions of the metering cell is determined based on the alignmentin the x and y dimensions of the fiducial marking. For example, themetering cell has a length and a width each about 2 μm. The fiducialmarking is placed substantially at the center of the metering cell. Inanother example, the fiducial marking is in the vicinity of or withinthe metering cell and their actual spatial relationship in the x and ydimensions does not change significantly from the design spatialrelationship.

At the process 6870, a focus score is determined and stored. In oneembodiment, the focus score is calculated based on the standarddeviation. In another embodiment, the focus score is calculated based onthe “edginess” of the image. For example, the “edginess” of the image isassessed by a sobel operator. In another example, the “edginess” of theimage is determined by an edge-sensitive computer program similar to ahigh pass filter. The techniques based on the “edginess” of the imageusually take into account that when the image is in sharp focus, highfrequency details are visible, and when the image is out of focus, thehigh frequency details are blurred or smudged. In yet anotherembodiment, the focus score is calculated based on histogram. Thehistogram techniques use specific characteristics of the fiducialmarking to improve focusing.

In yet another embodiment of the present invention, the images for thearea of interest are acquired by the imaging system. For each of atleast some of the acquired images, a first sobel square sum isdetermined. The sobel operator is applied to each data point on theacquired image. Each resultant value is squared, and all of the squaredvalues are added together. Additionally, the acquired image is blurred.For example, the blurring may be accomplished by applying Gaussiansmoothing to the acquired image. In one embodiment, the Gaussiansmoothing serves as a low pass filter attenuating high frequencycomponents of the acquired image. In another embodiment, the Gaussiansmoothing can be described as follows:

For the blurred image, a second sobel square sum is determined byapplying the sobel operator to the blurred image, squaring eachresultant value, and summing all the squared values. Afterwards,clipping is applied to the second sobel square sum. If the second sobelsquare is smaller than a predetermined threshold, the second sobelsquare sum is set to the predetermined threshold. Dividing the clippedsecond sobel square sum by the first sobel square sum, the resultantratio is used as the focus score. The focus score for each of at leastsome of the acquired images is then stored.

At the process 6880, the focus position for the metering cell isdetermined. As discussed above, at the process 6870, the focus scoresare obtained for various z positions. At the process 6880, in oneembodiment, the z position corresponding to a peak focus score is usedas the focus position. In another embodiment, the z positionscorresponding to two peak focus scores are determined and averaged. Theaverage z position is used as the focus position. In yet anotherembodiment, the focus position is determined based on the characteristicof the entire curve representing the focus score as a function of zposition.

In another embodiment, the fiducial marking has a known spatialrelationship with chambers within the metering cell in the z dimension.The focus position in the z dimension of the metering cell is determinedbased on the focus position in the z dimension of the fiducial marking.For example, the metering cell has a length and a width each about 2 μm.The fiducial marking is placed substantially at the center of themetering cell. In another example, the fiducial marking is in thevicinity of or within the metering cell and their actual spatialrelationship in the z dimension does not change significantly from thedesign spatial relationship.

FIG. 27 shows simplified curves for focus score as a function of zposition obtained at the process 6870 according to an embodiment of thepresent invention. The focus score at each z value is associated withthe sobel square sum for the acquired image without blurring. Thisdiagram is merely an example, which should not unduly limit the scope ofthe claims. One of ordinary skill in the art would recognize manyvariations, alternatives, and modifications. As shown in FIG. 27, focusscores are calculated at z-axis positions separated by approximately 2μm and extending for 100 μm on either side of z_(f). The coarse natureof the z-axis position determined by the process 4802 is evident, as thepeak of the focus score distributions are located approximately 20 μmfrom z_(f).

In another embodiment, the method by which the stage is scanned, thedensity of measurement points, and the range over which the measurementsextend can be varied, as would be evident to those skilled in the art.For example, focus scores are collected at fewer locations separated bygreater distances. In another example, focus scores collected at 10 μmspacing located on alternating sides of z_(f) is used as inputs to theimage processing software, only obtaining additional focus scores andfilling in the curve if needed.

FIG. 27 shows two different focus score runs in which the aperture ofthe condenser of the imaging system is operated in either a narrow or awide setting. A curve 5030 corresponds to a narrow setting andrepresents a bi-modal distribution of focus scores. The twin peaks areeach associated with the detection of the top and bottom edges of thefiducial marking, such as a company logo. This bi-modal distribution canbe characterized by a full width half magnitude (FWHM) 5035. If thecondenser aperture is operated at a wide setting, the bi-modaldistribution merges into a single peaked distribution represented by acurve 5020. The amplitude of the single peak is reduced from theamplitude characteristic of the bi-modal distribution and the FWHM isreduced as well. The FWHM of the single peak distribution is representedby line 5025.

Additionally, FIG. 28 shows simplified curves for focus score as afunction of z position obtained at the process 4804 according to oneembodiment of the present invention. The focus score at each z value isassociated with the sobel square sum for the acquired image withoutblurring. This diagram is merely an example, which should not undulylimit the scope of the claims. One of ordinary skill in the art wouldrecognize many variations, alternatives, and modifications. As shown inFIGS. 27 and 28, the focusing scores obtained without image blurring mayproduce irregular focal peaks under certain conditions. Sometimes thepeak is single modal, sometimes the peak is bi-modal, and usually thepeak is a combination of the two. Neither peak is guaranteed to be thetop peak and thus grabbing one peak over the other may result in a focusplane error on the order of tens of microns. If the depth of field ofthe imaging system is less than 10 microns, grabbing the wrong peak canproduce significantly out of focus images.

The disadvantage for obtaining focusing scores without image blurringcan be improved by blurring the image and calculating ratios asdiscussed above. FIG. 29 shows simplified curves for focus score as afunction of z position obtained at the process 4804 according to anotherembodiment of the present invention. This diagram is merely an example,which should not unduly limit the scope of the claims. One of ordinaryskill in the art would recognize many variations, alternatives, andmodifications. The focus score at each z value is associated with aratio that is taken between the sobel square sum for the acquired imageand the clipped sobel square sum for the blurred image. FIG. 28 and FIG.29 are produced from the same acquired images.

As shown in FIG. 29, the blurring and ratio technique in effectnormalizes the sobel output by the amount of that same output on ablurry version of the original. The peak of the curves in FIG. 29 occursfor the image which suffers the largest degradation as a result of theblurring operation. An image which suffers no degradation produces aratio value of 1.0. This normalization process can remove the dependencyof the sobel operation on the intensity of a particular image plane,which can fluctuate due to optical variations.

In certain embodiments, the number and scope of adjustments performed atthe process 4420 for alignment and focusing depend on the accuracy ofthe mapping from the design space to the measurement space at theprocess 4410. For example, bending or tilting of the microfluidicdevice, thereby shifting the metering cell out of the original plane ofthe microfluidic device, may result in additional z-axis focusingactions. These additional focusing steps may result in an increase inthe amount of time desired to acquire a high-resolution image of themetering cell. Improved mapping between the design space and measurementspace would enable the imaging system to move the metering cells toposition in which the acquisition of high-resolution images can beperformed with increased efficiency.

To further improve the mapping accuracy, in some embodiments, more thanthree fiducial markings may be used at the process 4410 to provide anon-planar transformation between the design space and the measurementspace. FIG. 30 is a simplified surface map of a three dimensionalflexible substrate according to an embodiment of the present invention.The warping or deformation of the microfluidic device is illustrated asan increase in z-axis height at certain x-y positions across theflexible substrate. In one embodiment, the inputs for this higher orderdimensional mapping could come from location information obtained usingmore than three fiducial markings. In another embodiment, inputs couldbe provided based on measurements made on the metering cell at theprocess 4420. Feedback from these measurements can be used to update andrefine the mapping as a function of time. Consequently, for anothermetering cell, placement of the microfluidic device in preparation forthe process 4420 would improve in accuracy as more data is obtained. Thegeneration of such a higher order dimensional mapping can substantiallyincrease the system throughput by reducing or even eliminating the needfor the process 4420 for some or all metering cells.

In one embodiment of the present invention, a 12 point microfluidicdevice registration process can be used that fits at least four fiducialmarkings with a non-planar surface. For example, a three dimensionalparabola could be used as the mapping surface. For example, the processof determining the coarse and fine locations of each fiducial markingcan contribute information used in calculation of the parabolic fittingparameters. In one embodiment, fiducials near the edges, the center, andother locations on the microfluidic device could be utilized, in variousorders, in the calculation of the parabolic fitting parameters. Inanother embodiment, the processes 4410 and 4420 could be combined into asingle predictive focus-based algorithm that uses higher order fittingand localized corrections to improve system throughput.

As discussed above, the method 4400 uses the processes 4410 and 4420 foralignment and focusing in certain embodiments. For example, at theprocess 4410, the alignment and focus of the fiducial marking associatedwith the metering cell are each within 100-μm accuracy. In anotherexample, at the process 4420, the alignment of the fiducial marking iswithin about 1-μm accuracy. In yet another example, at the process 4420,the focusing of the fiducial marking is within about 1-μm accuracy.

As shown in FIG. 18, at the process 4430, the metering cell is moved tothe focus position and an image of the metering cell is captured. Forexample, the captured image has a high resolution. In one embodiment,the image is acquired by the same camera that is used to capture thelow-resolution image at the process 4810. In another embodiment, a LeicaDC500 digital camera can be used to capture a high-resolution image. Forexample, the high resolution image has about 3900×3030 pixels and coversat least one well region including the fluid and species at a colordepth of 16 bits. In another example, the image includes the containmentlines, the wells, and the channels that connect the wells. In yetanother example, the metering cell is moved in the x dimension and/orthe y dimension in order to improve alignment prior to capturing theimage of the metering cell.

The captured image is then normalized. In one embodiment, the color andintensity of the acquired image is significantly affected by thecondition and operating voltage of the illumination source of theimaging system. For example, the illumination source is a bulb. As abulb ages, the overall hue of the image changes, with the red componentof the light increasing in intensity in comparison with the othercolors. This increase in red intensity may result from a decrease in thebulb temperature. Additionally, even with a constant illuminationsource, the opacity of the microfluidic device, which can depend onhydration levels and vary with time, may result in differences in imagebrightness. To correct for these artifacts and any radial vignettingintroduced by the microscope, a technique called image normalization canbe employed.

For image normalization, a calibration image is taken with themicrofluidic device removed from the imaging system with the stage at az calibration position. In one example, the z calibration position isdifferent from the focus position. The z calibration position may takeinto account changes to the illumination beam as the beam passes throughthe microfluidic device. In another example, the z calibration positionis the same as the focus position. The calibration image is then used tocorrect for the effects resulting from the condition and operatingvoltage of the illumination source. In one embodiment, the algorithmcalculates the ratio of the intensity of the acquired image of themetering cell to the calibration image on a pixel by pixel basis. Themicrofluidic device includes regions that contain substantially noinformation, the ratio of the intensities in these regions is set equalto unity. The intensity ratio is then multiplied by a scaling factor tomaximize the dynamic range around unity.

Although the mapping from this calibration image to the actual image maynot be linear due to the bending of light rays as they pass through themicrofluidic device and/or glass slab, the image normalizationeffectively white balances the image by adjusting the red, blue, andgreen components of the image. Additionally, the image normalizationimproves consistency between the attenuated edge pixels and the centerpixels. For example, the effects of white balance and consistencyimprovement are significant for low illumination conditions andparticular condenser and/or aperture settings in which the non-linearityis pronounced.

Moreover, the image is median shifted to move the centroid of the imagehistogram, i.e., counts as a function of intensity, to a known value.The image is also downgraded around that centroid to reduce the datasize in the image. For example, the intensity ratio is sampled at randomlocations on the microfluidic device. Using these sampled intensityratio values, the image is adjusted to shift the centroid of the imageto the known value. In one embodiment, the centroid is shifted to alignwith an intensity level of 128, and the image is downgraded to 8 bits.This shift may be used to either darken or brighten the image. In oneembodiment, the normalized, white balanced, and downgraded image isstored in a computer memory available for further processing.

As discussed above and further emphasized here, the above description ofthe process 4430 includes merely examples, which should not unduly limitthe scope of the claims. One of ordinary skill in the art wouldrecognize many variations, alternatives, and modifications. Although theabove has been shown using a selected sequence of processes, there canbe many alternatives, modifications, and variations. For example, someof the processes may be expanded and/or combined. Other processes may beinserted to those noted above. Depending upon the embodiment, thespecific sequences of processes may be interchanged with othersreplaced. Further details of these processes are found throughout thepresent specification.

For example, in another embodiment of the present invention, informationobtained at the process 4430 could be used as data inputs for theparabolic fitting at the process 4410 for another metering cell. In thisembodiment, the three dimensional locations of the metering cell, asdetermined from the high-resolution image, can provide informationuseful in determining the parabolic fitting parameters. For example, themetering cells near the center of the microfluidic device, separatedfrom the fiducial markings near the edges of the microfluidic device,may be measured earlier in time than metering cells near the fiducialmarkings. The early measurements of centrally located metering cells mayprovide for faster convergence of the fitting algorithm as the measuredlocation of these centrally located cells may differ from the planarmapping more than the measured locations of cells closer to the fiducialmarkings.

As discussed above, the method 4400 uses various fiducial markings invarious processes. In one embodiment, the fiducial markings can be anyphysical features associated with the microfluidic device. For example,the fiducial markings are on the handle substrate of the microfluidicdevice. In another example, the fiducial markings are on the flexiblesubstrate of the microfluidic device. The fiducial markings may includea channel wall or an edge of the microfluidic device. In yet anotherexample, the fiducials markings are selected from ones described inFIGS. 1-13A and 15A-15B.

Additionally, the method 4400 align and focus a metering cell andacquire an image of the metering cell. The alignment and focus processmay use at least one fiducial marking for the process 4420. The spatialrelationship between the fiducial marking and the metering cell does notchange significantly. For example, the fiducial marking is in thevicinity of the metering cell. The metering cell is merely an example,which should not unduly limit the scope of the claims. One of ordinaryskill in the art would recognize many variations, alternatives, andmodifications. In one embodiment, the method 4400 is applied to anyphysical feature on the microfluidic device. The physical feature isaligned and focused, and an image of the physical feature is taken. Forexample, the physical feature is a chamber. The chamber may be a well, afluid channel, a control channel, or else.

Moreover, the method 4400 may be performed by the imaging system 4010 oranother imaging system according to the instructions of the computersystem 4110 or another computer system. For example, a system forprocessing one or more microfluidic devices includes one or morecomputer-readable media and a stage for locating a flexible substrate.The flexible substrate comprises at least three fiducial markings, afirst additional fiducial marking, and a first chamber capable ofholding a fluid therein. For example, a volume of the fluid is less thana nanoliter. The one or more computer-readable media include one or moreinstructions for providing a flexible substrate, and one or moreinstructions for determining a transformation between a design space anda measurement space based on at least information associated with the atleast three fiducial markings. Additionally, the one or morecomputer-readable media include one or more instructions for performinga first alignment to the flexible substrate based on at leastinformation associated with the transformation between the design spaceand the measurement space, one or more instructions for acquiring atleast a first image of the first additional fiducial marking associatedwith the first chamber, one or more instructions for performing a secondalignment to the flexible substrate based on at least informationassociated with the first image, and one or more instructions foracquiring a second image of the first chamber associated with theflexible substrate.

The one or more instructions for determining a transformation between adesign space and a measurement space include one or more instructionsfor determining at least three actual locations corresponding to the atleast three fiducial markings respectively. The at least three fiducialmarkings are associated with at least three design locationsrespectively. Additionally, the one or more instructions for determininga transformation include one or more instructions for processinginformation associated with the at least three actual locations and theat least three design locations. The design space is associated with theat least three design locations and the measurement space is associatedwith the at least three actual locations. The one or more instructionsfor acquiring at least a first image of the first additional fiducialmarking include one or more instructions for acquiring a first pluralityof images of the first additional fiducial marking. The first pluralityof images includes the first image. Additionally, the one or moreinstructions for acquiring at least a first image includes one or moreinstructions for processing information associated with the firstplurality of images.

Moreover, the one or more computer-readable media includes one or moreinstructions for storing the second image in a memory. The memory is acomputer memory. The second image includes 3900 by 3030 pixels. Thesecond image comprises a 16 bit image. The one or more instructions forperforming a second alignment to the flexible substrate includes one ormore instructions for translating the flexible substrate in at least onedimension to position a chamber in preparation for capturing the secondimage. Also, the one or more computer-readable media includes one ormore instructions for normalizing the second image, one or moreinstructions for white balancing the second image, and one or moreinstructions for converting the second image from a first image depth toa second image depth. For example, the first image depth is 16 bits andthe second image depth is 8 bits.

In one embodiment, the first additional fiducial marking is a companylogo. The at least three fiducial markings include a company logo. Inanother embodiment, the flexible substrate is deformable in threedimensions. For example, the flexible substrate is deformed by actionsselected from the group consisting of fabrication, handling, andprotocols. The protocols can result in the flexible substrate swellingor contracting. In yet another embodiment, a relationship between thedesign space and the measurement space is non-planar. The flexiblesubstrate is deformed such that a planar transformation is capable toapproximately determine an actual location of the first chamber. In yetanother embodiment, the transformation between the design space and themeasurement space is non-planar. For example, the non-planartransformation comprises a three dimensional parabolic mapping. Thenon-planar transformation is updated using information obtained bycharacterization of a second additional fiducial marking.

Numerous benefits are achieved using the present invention overconventional techniques. Some embodiments provide at least one way toform alignment patterns for a deformable active region for amicrofluidic system. Certain embodiments rely on conventional materials,which are relatively easy to use. Some embodiments provide alignmentand/or focus based on mapping between the design space and themeasurement space. The transformation between the design space and themeasurement space uses, for example, at least three fiducial markings.Certain embodiments provide accurate focusing by acquiring and analyzinga plurality of images along at least one dimension. Some embodiments ofthe present invention perform alignment and focusing on a microfluidicdevice including at least one flexible substrate. The alignment andfocusing take into account the deformation of the flexible substrate.Certain embodiments improve throughput in imaging system. For example,the imaging system uses a computer system to automatically performalignment and focusing. In another example, mapping from the designspace to the measurement space increases the accuracy of stagepositioning, and thereby, the efficiency of high-resolution imageacquisition. Depending upon the embodiment, one or more of thesebenefits may exist. These and other benefits have been describedthroughout the present specification.

It is also understood that the examples and embodiments described hereinare for illustrative purposes only and that various modifications orchanges in light thereof will be suggested to persons skilled in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims.

1. A method for producing an image of an object within a chamber of amicrofluidic device comprising the steps of: providing said microfluidicdevice, said microfluidic device having x, y, and z dimensions andfurther comprising a chamber depth center point located between a topwall and a bottom wall of said chamber along said z dimension, saidchamber depth center point being located a known z dimension distancefrom an optically detectable fiducial marking embedded within saidmicrofluidic device at a z depth; placing said microfluidic devicewithin an imaging system comprising: an optical device capable ofdetecting said fiducial marking and transmitting said image of saidobject, said optical device defining an optical path axially alignedwith said z dimension of said microfluidic device and having a focalplane perpendicular to said optical path, wherein when said focal planeis moved along said optical path in line with said fiducial marking,said fiducial marking is maximally detected when said focal plane is atsaid z depth in comparison to when said focal plane is not substantiallyin-plane with said z depth, an image processing device in communicationwith said optical device, said image processing device being able tocontrol said optical device to cause said focal plane to move along saidz axis and move said focal plane to maximally detect said fiducialmarking, said image processing device being further able to transmitsaid image of said object; controlling said optical device with saidimage processing device to cause said focal plane to move along saidoptical path until said optical device maximally detects said fiducialmarking; further controlling said optical device with said imageprocessing device to move said focal plane along said optical path saidz dimension distance to cause said field depth center point to belocated at said chamber depth center point; and, imaging said objectwithin said chamber while said focal plane is located said chamber depthcenter point.
 2. The method of claim 1 wherein said microfluidic deviceis made wholly or partly from an elastomeric material.
 3. The method ofclaim 2 wherein said elastomeric material is polydimethlysiloxane. 4.The method of claim 1 wherein said microfluidic device is partly madefrom glass material.
 5. The method of claim 4 wherein said chamber isformed wholly or partly in said glass material.
 6. The method of claim 2wherein said chamber is wholly or partly within said elastomericmaterial.
 7. The method of claim 1 wherein said depth of field isgreater than, equal to, or less than the z dimension of said chamber. 8.The method of claim 1 wherein said optical device comprises an analogoutput charged coupled device type image detector and said imageprocessor comprises an analog to digital converter.
 9. The method ofclaim 1 wherein said optical device comprises a digital detectiondevice.
 10. The method of claim 1 wherein said image processing devicecomprises a digital computer and a data storage device.
 11. The methodof claim 10 wherein said digital computer comprises an output displayfor displaying said image of said object.
 12. A system for producing animage of an object within a chamber of a microfluidic device comprising:said microfluidic device, said microfluidic device having x, y, and zdimensions and further comprising a chamber depth center point locatedbetween a top wall and a bottom wall of said chamber along said zdimension, said chamber depth center point being located a known zdimension distance from a optically detectable fiducial marking embeddedwithin said microfluidic device at a z depth; an imaging system forplacing said microfluidic device therein comprising: an optical devicecapable of detecting said fiducial marking and transmitting said imageof said object, said optical device defining an optical path axiallyaligned with said z dimension of said microfluidic device and having afocal plane, wherein when said focal plane is moved along said opticalpath in line with said fiducial marking, said fiducial marking ismaximally detected when said focal plane is substantially in-plane withsaid z depth as compared to when said field depth center point is notsubstantially in-plane with said z depth, an image processing device incommunication with said optical device, said image processing devicebeing able to control said optical device to cause said focal plane tomove along said z axis and move said field depth center point tomaximally detect said fiducial marking, said image processing devicebeing further able to transmit said image of said object, said imageprocessing device being in operable communication with said opticaldevice to cause said focal plane to move along said optical path untilsaid optical device maximally detects said fiducial marking, whereinwhen said image processing device causes said optical device to movesaid focal plane along said optical path said z dimension distance, saidfocal point is located at said chamber depth center point.
 13. Thesystem of claim 12 wherein said microfluidic device is made wholly orpartly from an elastomeric material.
 14. The system of claim 13 whereinsaid elastomeric material is polydimethylsiloxane.
 15. The system ofclaim 12 wherein said microfluidic device is partly made from glassmaterial.
 16. The system of claim 12 wherein said chamber is formedwholly or partly in said glass material.
 17. The system of claim 13wherein said chamber is wholly or partly within said elastomericmaterial.
 18. The system of claim 12 wherein said depth of field isgreater than, equal to, or less than the z dimension of said chamber.19. The system of claim 12 wherein said optical device comprises ananalog output charged coupled device type image detector and said imageprocessor comprises an analog to digital converter.
 20. The system ofclaim 12 wherein said optical device comprises a digital detectiondevice.
 21. The system of claim 12 wherein said image processing devicecomprises a digital computer and a data storage device.
 22. The systemof claim 21 wherein said digital computer comprises an output displayfor displaying said image of said object.
 23. The system of claim 22wherein said output display comprises a graphical user interface.